Biochemical and Structural Basis for Inhibition of Enterococcus

Bingyin Peng , Manuel R. Plan , Panagiotis Chrysanthopoulos , Mark P. Hodson , Lars K. Nielsen , Claudia E. Vickers. Metabolic Engineering 2017 39, 20...
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Biochemical and Structural Basis for Inhibition of Enterococcus faecalis Hydroxymethylglutaryl-CoA Synthase, mvaS, by Hymeglusin D. Andrew Skaff,† Kasra X. Ramyar,‡ William J. McWhorter,‡ Michael L. Barta,‡ Brian V. Geisbrecht,*,‡ and Henry M. Miziorko*,† †

Division of Molecular Biology and Biochemistry and ‡Division of Cell Biology and Biophysics, University of MissouriKansas City, Kansas City, Missouri 64110, United States S Supporting Information *

ABSTRACT: Hymeglusin (1233A, F244, L-659-699) is established as a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme−inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS−hymeglusin cocrystal structure (1.95 Å) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.

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results showed that the inhibitor’s extended aliphatic chain bound in a funnel-shaped cavity of this plant enzyme and confirmed the formation of a thioester between the inhibitor’s β-lactone and the active site cysteine. While hymeglusin was originally described as an antibiotic, little is known about its interaction with mvaS, the bacterial HMG-CoA synthase. This report characterizes the inhibitor’s effect on Enterococcus faecalis and on isolated E. faecalis mvaS. Differences between the persistence of the inhibition observed for bacterial cells in light of analogous observations documented for animal cells7 prompted comparison of the enzyme−inactivator adducts for E. faecalis and human enzymes. The results suggested possible differences in the hymeglusin binding sites. Crystallization of hymeglusin-inactivated mvaS allowed a test of this hypothesis. The complementary biochemical and structural results of these studies are presented.

ymeglusin {(2E,4E,7R)-11-[(2R,3R)-3-(hydroxymethyl)4-oxooxetan-2-yl]-3,5,7-trimethylundeca-2,4-dienoic acid} was chemically characterized by Aldridge et al.1 and described as an antibiotic, but its biological properties were not reported. Tomoda et al.2 subsequently demonstrated hymeglusin’s antimicrobial activity against several fungi and bacteria.

After Miziorko and Behnke3,4 identified 3-chloropropionyl-CoA as a mechanism-based inactivator of animal HMG-CoA synthase (EC 4.1.3.5), hymeglusin was identified as the first natural product that specifically inhibits this cholesterogenic enzyme.5,6 This led to an expanded effort in characterizing hymeglusin inhibition of eukaryotic cholesterol biosynthesis6,7 and of eukaryotic HMG-CoA synthase.8 Both 3-chloropropionyl-CoA and hymeglusin target the active site cysteine involved in forming the two reaction intermediates, which are thioester adducts of enzyme to acetyl or HMG-CoA groups (Schemes 1 and 2). The confirmation of the active site function of the cysteine targeted by these inactivators was provided by the demonstration9 that alanine or serine substitution of this residue produced an enzyme that was devoid of catalytic activity and unable to form the covalent reaction intermediates. More recently, Pojer et al.10 reported the structure of an adduct of Brassica juncea HMG-CoA synthase with hymeglusin. These © XXXX American Chemical Society

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EXPERIMENTAL PROCEDURES Materials. Acetyl-CoA and acetoacetyl-CoA were synthesized using acetic anhydride and diketene, respectively, according to the procedure of Simon and Shemin. 11 Hymeglusin (L-659-699) was a generous gift from M. D. Greenspan (Merck Research Laboratories). Hydroxylamine hydrochloride was purchased from Eastman Laboratory Received: January 10, 2012 Revised: April 16, 2012

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Scheme 1. HMG-CoA Synthase (mvaS) Reaction

Scheme 2. Reaction of Hymeglusin with the Active Site Cysteine of HMG-CoA Synthase (mvaS)

media, containing either 0 or 25 μM hymeglusin, were inoculated with 200 μL of an overnight culture of E. faecalis. Samples were incubated with shaking for 3 h at 37 °C. A 3 mL aliquot of each culture was centrifuged at 3000g for 5 min to pellet bacteria before resuspension in either 3 mL of fresh LB or fresh LB containing 25 μM hymeglusin. At 30 min intervals the absorbance of each culture was measured at 600 nm. Kinetic Characterization of Inhibition of E. faecalis mvaS by Hymeglusin. Enzyme activity was measured at 412 nm by the DTNB method of Skaff and Miziorko.14 Purified mvaS (48 nM) was incubated with hymeglusin (75−600 nM) in 100 mM Tris-HCl (pH 8.0). The reaction was performed at 18 °C to allow measurement of activity at an adequate number of time points while maintaining elevated hymeglusin/enzyme concentration ratios. At the specified time points, 400 μM acetyl-CoA (approximately Km level) was added to the incubation mix to acetylate the free enzyme and protect against further formation of any hymeglusin adduct. Acetoacetyl-CoA (7 μM) was then added to initiate the measurement of enzyme activity, which was performed in the presence of 0.2 mM DTNB. The data, which indicated time-dependent loss of activity, were fit to semilog plots of percent residual activity versus time using a linear model and Microsoft Excel; correlation coefficients ranged from 0.970 to 0.995. Nonlinear regression fits (GraphPad Prism 4) of the data indicated a kinact of 3.5 ± 0.6 min−1 and a KI of 700 ± 18.5 nM. Recovery of HMGCS and mvaS Activity from Hymeglusin Inhibition. Purified human HMGCS14 or E. faecalis mvaS samples (9 μM) were incubated with 20 μM hymeglusin at room temperature in 0.1 M Tris-HCl (pH 8.0) for 1 h. After this incubation period, each protein sample retained 95% inhibition achieved at extended incubation times. These results suggest hymeglusin inactivation of the enzyme by formation of a covalent adduct between the inhibitor and protein, as observed with the animal enzyme. Inactivation follows first-order kinetics, and the doublereciprocal replot of apparent kinact values as a function of hymeglusin concentration indicates saturable binding and provides estimates of a KI of 606 nM and a kinact of 2.75 min−1 (inset of Figure 2 and Table 2). Nonlinear regression analysis of the primary data indicates a KI of 700 ± 18.5 nM and a kinact of 3.5 ± 0.6 min−1 (Table 2). A partition ratio of 11.7 ± 0.6 characterizes inactivation of mvaS by hymeglusin. In comparison, inhibition of human HMG-CoA synthase by hymeglusin is characterized by a KI of 53.7 nM and a kinact of 1.06 min−1.8 Recovery of the Activity of Hymeglusin-Inactivated mvaS and Human HMG-CoA Synthase upon Treatment with Hydroxylamine. Neutralized hydroxylamine disrupts the thioester adducts of the HMG-CoA synthase reaction intermediates in which the acyl groups of acetyl-CoA or HMG-CoA are linked to the active site cysteine.22 In hymeglusin-inactivated E. faecalis mvaS or human HMG-CoA synthase, any thioester adduct formed between the β-lactone inhibitor and the active site cysteine should also be disrupted if it is accessible to an aqueous solution of hydroxylamine. When the resulting hydroxamic acid derivative of hymeglusin is released from the active site cavity, enzyme activity could, in principle, be restored. This possibility was tested by incubation of hymeglusin-inactivated bacterial and human enzymes with 1 mM hydroxylamine. Substantial activity was restored at a rate for the human enzyme (0.0097 ± 0.0001 milliunit/min) that was 8-fold higher than that measured for the E. faecalis enzyme (0.0012 ± 0.0002 milliunit/min) (Table 2). These results are compatible with the hypothesis that, for E. faecalis mvaS, the enzyme−S−hymeglusin thioester adduct is less accessible to hydroxylamine in aqueous solvent than the corresponding adduct formed using human HMG-CoA synthase.

Figure 1. Recovery of the cellular growth of E. faecalis after removal of hymeglusin. E. faecalis cells were grown in LB for 3 h at 37 °C in the presence or absence of 25 μM hymeglusin. Cells were pelleted, then resuspended in fresh LB, and again incubated at 37 °C. The absorbance was measured at 600 nm. The traces correspond to LB prior to cell pelleting followed by post-resuspension growth in LB (■), hymeglusin and LB followed by post-resuspension LB (▲), and hymeglusin and LB followed by post-resuspension hymeglusin and LB (▼). The individual time points are averages of data from three experiments performed in parallel. Curves represent nonlinear regression fits of the data to a sigmoidal equation (GraphPad Prism 4). Inflection times and error estimates are listed in Table 2.

fresh LB in the absence of an inhibitor. These cells reached a growth curve inflection point at 3.1 h (Figure 1 and Table 2). The observations indicate that the putative adduct of hymeglusin to mvaS can slowly be hydrolyzed to restore enzyme activity and the pool of essential polyisoprenoid metabolites. In contrast with these observations for E. faecalis, animal cells require only 1 h to exhibit a 50% rebound from inhibition.7 Such observations would be compatible with the hypothesis of different hydrolysis rates for inhibitor adducts to animal HMG-CoA synthase versus bacterial mvaS. Time-Dependent Hymeglusin Inactivation of E. faecalis mvaS. Ambiguities in the nature of hymeglusin inhibition of animal HMG-CoA synthase6,7 were resolved by the results of Rokosz et al.8 Work with purified recombinant human HMG-CoA synthase clearly indicated time-dependent inactivation. In contrast, the nature of hymeglusin inhibition of bacterial mvaS has not been studied in detail. A basic kinetic characterization of purified E. faecalis mvaS [Vm = 10 units/mg (corresponding to kcat = 6.9 s−1); KmAcCoA = 350 μM; KmAcAcCoA = 10 μM] has been reported previously,21 but the scope of that

Table 2. Hymeglusin Inhibition of E. faecalis and Human HMG-CoA synthasea sample or enzyme E. faecalis cells E. faecalis cells with hymeglusin E. faecalis mvaS nonlinear fit method

graphical method

human HMGCSb

a

time to growth curve inflection point (h)

inactivation kinetic parameters

rate of activity restoration by 1 mM hydroxylamine (milliunit of activity recovered/min)

0.71 ± 0.03 3.05 ± 0.04

KI = 700 ± 18.5 nM kinact = 3.5 ± 0.6 min−1 kinact/KI = 0.0050 ± 0.0016 nM−1 min−1 KI = 606 nM kinact = 2.75 min−1 kinact/KI = 0.0045 nM−1 min−1 KI = 53.7 nM kinact = 1.06 min−1 kinact/KI = 0.0197 nM−1 min−1

0.0012 ± 0.0002

0.0097 ± 0.0001

Experimental details are provided in the legends of Figures 1 and 2 and Experimental Procedures. bValues reported by Roskosz et al.8 D

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Figure 2. Time-dependent inactivation of E. faecalis HMG-CoA synthase/mvaS by hymeglusin. Hymeglusin was incubated with HMGCS/mvaS protein for the indicated times prior to addition of acetyl-CoA and DTNB. After a baseline level of 412 nm absorbance had been recorded, acetoacetyl-CoA was added to initiate the activity assay. Reaction mixtures contained 100 mM Tris-HCl (pH 8.0), 0.2 mM DTNB, 400 μM Ac-CoA, 7 μM acetoacetyl-CoA, and 2 μg/mL enzyme. A semilog plot of percent HMGCS activity vs time is shown. Hymeglusin concentrations of 75 (■), 150 (◆), 225 (▲), 300 (□), and 600 nM (◇) were used. Progress curves were fit to a linear model with R2 values of >0.97. The inset is a replot of the reciprocal of the apparent kobserved value (derived from t1/2 values of data sets depicted in the main figure) vs the reciprocal of hymeglusin concentration. Intercepts on the x and y axes were used to determine a KI of 606 nM and a kinact of 2.75 min−1, respectively. Nonlinear regression fits to the primary data were also performed (GraphPad Prism 4) and give a KI of 700 ± 18.5 nM and a kinact of 3.5 ± 0.6 min−1 (Table 2).

Crystallographic Analysis of Hymeglusin Bound to mvaS. To further test this hypothesis, attempts were made to determine the structure of the hymeglusin−mvaS adduct, which would provide additional information about the access of solvent to the active site cysteine when the hymeglusin aliphatic chain occupies the active site cavity. Previous studies have established crystallization conditions for E. faecalis mvaS, which were used at that time to determine its structure both free and bound to the substrate acetoacetyl-CoA.17 Although initial attempts to reproduce these crystals with the enzyme preparation described here were unsuccessful, an alternative crystal form was identified (Experimental Procedures). This novel crystal form was characterized by a change to a lowersymmetry space group (from I222 to P21) but with an increase in apparent resolution (from 2.4 to 1.6 Å) for the unbound enzyme. Using these crystals, a structure of mvaS was determined and refined to Rwork and Rfree values of 14.1 and 19.4%, respectively. This structure consists of four crystallographically unique copies of the enzyme within the asymmetric unit (Figure S1 of the Supporting Information and Table 1). By contrast, the previously determined structure of the same enzyme contained two polypeptides within its asymmetric unit.17 Diffraction quality samples were also obtained from E. faecalis mvaS that had been briefly incubated with an equimolar concentration of hymeglusin immediately prior to establishment of the crystallization drops. These crystals of hymeglusinbound mvaS displayed only minor changes in cell constants when compared to those of the unbound enzyme and diffracted synchrotron X-rays to 1.95 Å limiting resolution (Table 1). After the collection of a nearly complete data set, the corresponding structure was determined by molecular replace-

ment and refined to Rwork and Rfree values of 17.1 and 21.8%, respectively (Figure 3 and Table 1). Examination of both 2Fo − Fc and Fo − Fc difference electron density maps following initial rounds of refinement revealed the presence of largely contiguous, unmodeled density within the active site cavity for all four crystallographically unique copies of mvaS present within the asymmetric unit (Figure S2 of the Supporting Information). The extended nature of this density was consistent with the branched, aliphatic tail group of hymeglusin. Furthermore, it was contiguous with the active site Cys111 side chain in all four copies; this was indicative of a covalent adduct that would be anticipated following hymeglusin modification of mvaS. The readily interpretable nature of this unmodeled density allowed accurate placement of a single hymeglusin ligand in each active site (Figure 3A). Comparison of the hymeglusin conformations in all four active sites revealed that the ligand bound in essentially identical orientations with respect to mvaS (Figure 3B and Figures S2 and S3 of the Supporting Information). In particular, superposition of all four hymeglusin chains within the refined structure yielded an average overall rmsd value of 0.72 Å across the entire ligand molecule. Interestingly, whereas the regions of the hymeglusin molecules involved in covalent linkages to the Cys111 thiolates superimpose quite well with one another, areas of greater conformational variability between the ligands bound at different mvaS active sites are found in the distal regions of the inhibitor. This is most likely because this region of hymeglusin is quasi-linear and aliphatic in nature and (aside from the terminal carboxylate) lacks hydrogen bond donors and acceptors or other noteworthy structural features that E

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Figure 3. 1.95 Å structure of E. faecalis mvaS covalently modified by hymeglusin. Crystals of E. faecalis mvaS that had been briefly incubated with hymeglusin were produced as described in Experimental Procedures and used to collect monochromatic diffraction data. Following structure solution by molecular replacement, electron density maps that allowed modeling of bound ligand in each of the four crystallographically independent active sites were calculated. (A) Stereoview of the mvaS active site for chain A. The protein backbone is depicted in cartoon format (purple), while the side chains of Glu79, Tyr143, and His233 are shown as cyan sticks. The catalytic Cys111 is colored green, and carbon atoms of covalently bound hymeglusin are colored yellow. The 2Fo − Fc map (blue mesh at 1.1σ contour) of the refined structure corresponding to the Cys111 thiolate− hymeglusin adduct is also shown. (B) Overlay of hymeglusin molecules as modeled in chains A−D, colored blue, green, orange, and red, respectively. The position of C8, which forms a covalent bond with the thiolate sulfur derived from Cys111, is denoted with an arrow. Additional information for all four active sites can be found in Figures S2−S4 of the Supporting Information.

Altered Stability of Their Hymeglusin Adducts. While Ef mvaS and the human and plant synthases are only approximately 27 and 28% identical, respectively, their overall folds are quite similar (Figure 4A), and all active site residues with clearly defined function are conserved. This conservation of mechanism is reflected in the analogous pairs of interactions formed between key enzyme side chains and the polar groups of the inhibitor hymeglusin, as mentioned above. Nevertheless, the conserved nature of the active site residues provided no insight regarding the differences in kinetic inactivation or hydroxylamine reactivation of mvaS versus its eukaryotic counterpart (Table 2). This suggested that regions at the active site periphery instead may be responsible for these differences. Indeed, an immediate and striking difference between the Ef mvaS and eukaryotic (B. juncea) HMG-CoA synthase involves the nature of the entrances to their respective active site cavities (Figure 4). The plant enzyme is characterized by a relatively wide cavity that is reminiscent of a triangular or pyramidalshaped funnel, with the bound hymeglusin laying flat against one its internal faces and extending nearly 16 Å into the catalytic core. For the purposes of illustration, the Cβ atom of Lys256 serves as a convenient landmark for defining boundaries of the active site funnel, because it lies only 3.2 Å from the hymeglusin carboxylate. Using this as a guide, the approximate dimensions of the funnel opening are 15.6 Å (Lys256 to Cγ1 of Val204), 17.2 Å (Lys256 to Cβ of Lys34), and 20.2 Å (Lys256 to Cβ of Pro155). It is particularly notable that the catalytic cysteine (Cys117), which participates in the thioester bond that

might otherwise significantly restrict its conformation toward the entrance of the mvaS catalytic cleft. The tubelike nature of the Ef mvaS active site channel allows nearly the entire hymeglusin molecule to be buried within the cavity upon binding. On average, 381.4 Å2 of surface area are buried through formation of the enzyme−inhibitor complex. This corresponds to approximately 84% of the total surface area available within the hymeglusin molecule. Despite the fact that a large portion of hymeglusin is aliphatic, two key polar interactions between its various functional groups and mvaS side chains stand out (Figure S4 of the Supporting Information). These are hydrogen bonds between the side chain Nε2 atom of His233 and the O6 alcohol group of hymeglusin (2.6 Å distance), and the side chain carboxylate of Glu79 and the O5 alcohol of the inhibitor (2.9 Å distance). Previous kinetic and structural studies have consistently suggested that Glu79 functions as a general acid/base during catalysis.17,22−24 His233 has also been assigned a role as a general acid/base during the condensation partial reaction of HMGCoA synthesis;24 however, it is also worth noting this residue contributes to binding of the second substrate, AcAc-CoA, by forming hydrogen bonds to its acetoacetyl group.22−24 Similar interactions have also been described for His247 and Glu48 of the B. juncea HMG-CoA synthase in its hymeglusin-bound state.10 This further emphasizes the highly conserved nature of these specific side chains and their contributions to HMG-CoA synthase function across diverse evolutionary lineages. Differences in Active Site Accessibility between the Eukaryotic and Prokaryotic Synthases Explain the F

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and animal origin. While inhibition of human HMG-CoA synthase by this fungal polyketide has been studied in some detail,8 relatively little information is available regarding the specific nature of hymeglusin’s effects on the bacterial synthase. This prompted us to conduct our study, which investigated the kinetic parameters of inactivation of mvaS by hymeglusin both in live E. faecalis cells and in vitro. Despite a common mode of inactivation (i.e., covalent modification), we observed significant differences in a number of biochemical parameters between the human and bacterial synthases (Table 2). The human synthase is far more sensitive to inhibition by hymeglusin, as demonstrated by its 10-fold lower KI value (53.7 nM) relative to that of mvaS (700 nM). This differential affinity influences the inactivation efficiency (kinact/KI), which is higher for human HMG-CoA synthase (0.0197) than for E. faecalis mvaS (0.0050) (Table 2). However, significant differences between both the formation and the stability of hymeglusin−synthase adducts were seen. Hymeglusin-mediated inactivation manifests itself at a faster rate in bacterial mvaS than in human HMG-CoA synthase. Inactivation is also more transient (shorter in duration) for the human enzyme than for its bacterial counterpart. Apparent differences in the duration of inactivation are in accord with the approximately 8-fold slower activity recovery rate following hydroxylamine treatment for inhibited E. faecalis mvaS when compared to human HMGCoA synthase. This strongly suggested that differences in active site solvent accessibility contributed to the differences between inactivation of the human and bacterial enzymes. We obtained further insight into the basis for hymeglusin inhibition of E. faecalis mvaS through structural studies of the covalently modified enzyme. In doing so, a novel crystal form of E. faecalis mvaS was identified that provided improved diffraction limits when compared to that previously reported for the same enzyme.17 Interestingly, the unit cell parameters for the crystal system described here match exceedingly well those reported by Campobasso and colleagues for Staphylococcus aureus mvaS (which is ∼60% identical) when bound to its substrate acetoacetyl-CoA.23 Comparison of the hymeglusin-bound E. faecalis mvaS structure presented here to those of either the E. faecalis17 or S. aureus23 enzyme bound to various CoA derivatives reveals that the aliphatic tail of hymeglusin occupies a region within the enzymes’ relatively long, active site tunnel that is largely identical to that of the pantothenic acid moiety of CoA in these closely related structures (Figure 5). That said, it is noteworthy that the hymeglusin tail is completely extended and not kinked as in the distal, reactive region of CoA. This elongated conformation allows the covalent inhibitor to effectively bypass the hydrophobic crevice formed by Tyr205, Met239, and Tyr306 of mvaS, which itself accommodates the reactive cysteine/thiolcontaining functionality of the coenzyme. Intriguingly, this stands in contrast to what is observed for the B. juncea HMGCoA synthase when it is bound to hymeglusin, acetyl-CoA, or HMG-CoA.10 Here, the aliphatic hymeglusin chain actually follows, rather than bypasses, the kink found in the distal region of the CoA (Figure 6). In doing so, the terminal region of the hymeglusin chain binds to an opposing face of the funnelshaped entrance to the enzyme’s active site versus what is seen for either CoA derivative. Such a dramatic difference in ligand binding mode is not possible within the bacterial enzyme, as its tunnel-shaped active site entrance imposes significant structural restraints on any inhibitors or substrates that occupy this region. One important consequence of these architectural

Figure 4. Comparison of hymeglusin-bound structures of eukaryotic HMG-CoA synthase and bacterial mvaS. (A) Crystal structure of hymeglusin-bound B. juncea HMG-CoA synthase (PDB entry 2F9A, orange cartoon) superimposed with that of hymeglusin-bound E. faecalis mvaS (PDB entry 3V4X, purple cartoon). Positions of the respective catalytic cysteine residues are colored green. (B) B. juncea HMG-CoA synthase shown as a molecular surface in an orientation identical to that in panel A. Bound hymeglusin is shown as a yellow ball and stick. The positions of landmark residues that define the boundaries of the active site entrance are indicated and colored purple. (C) E. faecalis mvaS shown as a molecular surface in an orientation identical to that in panel A. Bound hymeglusin is shown as a yellow ball and stick. The positions of landmark residues that define the boundaries of the active site entrance are indicated and colored orange.

covalently traps the inhibitor in place, has otherwise unrestricted access to the bulk solvent via the unoccupied side of this funnel. In contrast, the Ef mvaS entrance is far more restricted and resembles a tunnel or tube. In this case, the backbone O of Gly148 can be used as a landmark as it lies only 4.8 Å from the hymeglusin carboxylate. While this tunnel extends nearly 16 Å into the catalytic core, its opening is only 9.4 Å in diameter (as measured from Gly148 to the side chain amide of Asn202). Because of this, covalently linked hymeglusin appears to be capable of quite effectively occluding the access of bulk solvent to the Ef mvaS catalytic Cys111. Thus, consideration of these architectural differences at the respective active site peripheries provides a structural explanation for the ∼8-fold slower recovery of activity upon hydroxylamine treatment for mvaS relative to the eukaryotic synthase (Table 2).

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DISCUSSION Hymeglusin has been recognized and described as a potent, covalent inhibitor of HMG-CoA synthases from bacterial, plant, G

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Figure 5. Comparison of E. faecalis mvaS structures bound to either acetoacetate and CoA or hymeglusin. (A) Chain B from the structure of mvaS bound to acetoacetate and CoA (PDB entry 1YSL). The polypeptide is shown as a purple cartoon, while the ligands are shown in ball and stick format. The catalytic cysteine residue is colored green. (B) Chain A from the structure of mvaS bound to hymeglusin (PDB entry 3V4X). (C) Superposition of the CoA and hymeglusin structures as they lie within the mvaS active site. Note that the alphatic tail of hymeglusin largely occupies the same position as the pantothenic acid moiety of CoA. The positions of acetoacetate (AcAc), coenzyme A (CoA), and C8 of hymeglusin (C8) are denoted with arrows.

differences among HMG-CoA synthases across various kingdoms of life is that the active sites of the eukaryotic forms appear to have relatively free access to bulk solvent, whereas their bacterial counterparts are more restricted. While the physiological relevance of this difference is not clear at this time, it does provide a rational basis for the notably altered kinetic inactivation parameters reported here (Figures 1 and 2 and Table 2) and elsewhere.8 Furthermore, it also strongly suggests that inhibitors that are highly selective for the bacterial enzymes over their human homologue can be discovered, optimized, and developed. In the description of the eukaryotic HMGCS−hymeglusin adduct structure,10 it was noted that the active site glutamate hydrogen bonds to the C2 hydroxyl of the ring-opened inactivator. On the basis of such structural observations, additional function for the catalytic glutamate in catalysis of thioester hydrolysis was proposed. Such a hypothesis can be discounted on the basis of results of direct functional tests of the thioester hydrolysis partial reaction.22 Alanine substitution of the catalytic glutamate produces a mutant enzyme that is unimpaired in catalysis of this partial reaction. Neither Vm nor Km values are substantially different from those of the wild-type enzyme. In contrast, however, this mutant exhibits a >5 orders of magnitude diminution of the overall reaction (Scheme 1) in comparison with the wild-type enzyme. This confirms the previous functional assignment of this active site glutamate in the condensation partial reaction.22,24 In addition to hymeglusin’s ability to inactivate both prokaryotic and eukaryotic HMG-CoA synthase, we recently demonstrated the ability of epoxide-containing molecules to inhibit the eukaryotic enzyme.25 Cerulenin is an antibiotic that inhibits β-ketoacyl acyl carrier protein synthases, the bacterial fatty acid-condensing enzymes.26 Its epoxide moiety reacts with the active site cysteine to form a stable thioether adduct. These enzymes as well as HMG-CoA synthases are members of the family of initial condensation enzymes, and they exhibit

Figure 6. Comparison of available ligand-bound structures for E. faecalis and S. aureus mvaS and B. juncea HMG-CoA synthases. (A) Superposition of enzyme monomers from E. faecalis mvaS bound to hymeglusin (PDB entry 2V4X) and acetyl-CoA (PDB entry 1YSL, chain B), S. aureus mvaS bound to acetoacetyl-CoA (PDB entry 1TXT, chain A), and B. juncea HMG-CoA synthase bound to hymeglusin (PDB entry 2F9A), acetyl-CoA (PDB entry 2FA3), and HMG-CoA (PDB entry 2FA0). The E. faecalis and S. aureus structures are colored purple and blue, respectively, while the B. juncea structures are colored orange. The location of the helical insertion domain that is found only in the eukaryotic enzyme is highlighted with a dashed green circle. The positions of the bound ligands and inhibitors are shown in ball and stick format. Note that each of these ligands or inhibitors adopts the same positional conformation, except for hymeglusin when bound to B. juncea HMG-CoA synthase. Instead, this inhibitor binds to an opposing face of the active site funnel when compared to the CoA derivatives. (B) Magnified views of the positions of each of the ligands and inhibitors from panel A as they lie within the superimposed enzyme active sites. All CoA-derived ligands are shown colored with their carbon atoms in yellow, while hymeglusin as found in the E. faecalis and B. juncea enzymes is colored with purple and orange carbon atoms, respectively.

considerable structural and functional homology. On this basis, it seemed reasonable to test cerulenin against HMG-CoA synthase; the human enzyme proves to be sensitive to this inhibitor, displaying time-dependent inactivation.25 S. aureus mvaS also exhibits time-dependent inactivation by cerulenin (I. Misra and H. M. Miziorko, unpublished results). Ceestatin, a novel epoxide-containing compound, inhibits hepatitis C virus replication via a mechanism attributed to its demonstrated time-dependent inactivation of eukaryotic (human) HMG-CoA synthase.25 While these epoxide inhibitors are effective, in vitro, at concentrations higher than those required for hymeglusin, the thioether adducts that presumably form upon active site H

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acid; Ef , E. faecalis; LB, Luria-Bertani medium; rmsd, rootmean-square deviation.

cysteine modification are not labile under the same physiological conditions that permit thioester adduct hydrolysis. This is particularly relevant in terms of potential antiinfective applications of hymeglusin and related compounds, because data presented here show that hymeglusin’s effect on live bacterial cells is reversible on the order of hours (Figure 1 and Table 2). Thus, in any future work on the design or optimization of HMG-CoA synthase inhibitors, the chemical nature of the protein−inhibitor adduct should receive consideration. In this regard, the structural information available on enzyme−inhibitor complexes along with the characterized differences in kinetic inactivation parameters presented herein should prove to be a valuable asset moving forward.

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ASSOCIATED CONTENT

S Supporting Information *

Four supplemental figures and their figure legends. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*H.M.M.: e-mail, [email protected]. B.V.G.: e-mail, [email protected]; School of Biological Sciences, University of MissouriKansas City, 5100 Rockhill Rd., Kansas City, MO 64110; telephone, (816) 235-2246; fax, (816) 2355595. Author Contributions

D.A.S. and K.X.R. contributed equally to this work. Author Contributions

B.V.G. and H.M.M. shared supervision of this work. Funding

This work was supported in part by National Institutes of Health Grants AI071028 and AI090149 and the MarionMerrell-Dow Foundation. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Michael Greenspan (Merck) generously provided the hymeglusin used in these studies. Dr. Ila Misra (Medical College of Wisconsin) determined that hymeglusin is a timedependent inactivator of S. aureus mvaS. We acknowledge generous technical assistance of Drs. Rod Salazar, Andy Howard, and John Chrzas during X-ray diffraction data collection. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, via Contract W-31-109-Eng-38. Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) beamlines at the Advanced Photon Source, Argonne National Laboratory. A list of supporting member institutions may be found at http://www.ser-cat.org/members. html.

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ABBREVIATIONS HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; Ac-CoA, acetylCoA; AcAc-CoA, acetoacetyl-CoA; F244 (or 1233A), hymeglusin; mvaS, prokaryotic HMG-CoA synthase; HMGCS, eukaryotic HMG-CoA synthase; DTNB, dithiobisnitrobenzoic I

dx.doi.org/10.1021/bi300037k | Biochemistry XXXX, XXX, XXX−XXX

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